1. Field of the Invention
The present invention relates to the microbiological industry, and specifically to a method for producing an aromatic L-amino acid using a bacterium of the Enterobacteriaceae family which has been modified to attenuate expression of the ydiN gene, the ydiB gene, or both.
2. Brief Description of the Related Art
Conventionally, L-amino acids are industrially produced by fermentation utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including by transforming microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042, or U.S. Pat. Nos. 4,346,170, 5,661,012, and 6,040,160).
Another way to enhance L-amino acid production yields is to attenuate expression of a gene or several genes involved in degradation of the target L-amino acid, genes which divert the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, genes involved in the redistribution of carbon, nitrogen, and phosphate fluxes, genes coding for toxins, etc.
Shikimate dehydrogenase catalyzes the fourth step of the shikimate pathway, which is the essential route for the biosynthesis of aromatic compounds in plants and microorganisms. Escherichia coli expresses two shikimate dehydrogenase paralogs, the NADP-specific AroE and a putative enzyme YdiB. YdiB is characterized as a dual specificity quinate/shikimate dehydrogenase that utilizes either NAD or NADP as a cofactor. The structures of AroE and YdiB with bound cofactors were determined at 1.5 and 2.5 A resolution, respectively. Both enzymes display a similar structure with two alpha/beta domains separated by a wide cleft. Comparison of their dinucleotide-binding domains reveals the molecular basis for cofactor specificity. Independent molecules display conformational flexibility which suggests that a switch between the open and closed conformation occurs upon substrate binding. Sequence analysis and structural comparison led to a proposal for the catalytic machinery and a model for 3-dehydroshikimate recognition. (Michel G., et al., J Biol. Chem. 23; 278(21):19463-72 (2003)).
The Escherichia coli YdiB protein, an orthologue of shikimate 5-dehydrogenase, catalyzes the reduction of 3-dehydroshikimate to shikimate as part of the shikimate pathway, which is absent in mammals but required for the de novo synthesis of aromatic amino acids, quinones, and folate in many other organisms. In this context, the shikimate pathway has been selected as a target for the development of antimicrobial agents. The crystal structure of YdiB shows that the promoter contains two alpha/beta domains connected by two alpha-helices, with the N-terminal domain being novel and the C-terminal domain being a Rossmann fold. The NAD+ cofactor, which co-purified with the enzyme, is bound to the Rossmann domain in an elongated fashion with the nicotinamide ring in the pro-R conformation. Its binding site contains several unusual features, including a cysteine residue opposite to the nicotinamide ring and a clamp-like structure over the ribose of the adenosine moiety formed by phenylalanine and lysine residues. The structure explains the specificity for NAD versus NADP in different members of the shikimate dehydrogenase family on the basis of variations in the amino acid identity of several other residues in the vicinity of this ribose group. A cavity lined by residues that are 100% conserved among all shikimate dehydrogenases is found between the two domains of YdiB, in close proximity to the hydride acceptor site on the nicotinamide ring. Shikimate was modeled into this site in a geometry such that all of its heteroatoms form high quality hydrogen bonds with these invariant residues. Their strong conservation among all the orthologues supports the possibility of developing broad spectrum inhibitors of this enzyme. The nature and disposition of the active site residues suggest a novel reaction mechanism in which an aspartate acts as the general acid/base catalyst during the hydride transfer reaction (Benach J., et. al., J Biol. Chem. 23; 278(21):19176-82 (2003)).
When shikimic acid is produced by genetically modified Escherichia coli, it has previously been found that carbon-rich conditions (e.g. phosphate-limiting) favor production of shikimic acid over shikimate pathway by-products, whereas the situation is the opposite under carbon-(glucose-) limited conditions. Gene expression patterns of the shikimate producing strain W3110.shik1 (W3110 with an aroL deletion and plasmid-overexpressed aroF) and the wild-type strain W3110 grown under carbon- and phosphate-limited (carbon-rich) chemostat conditions (D=0.23 h(−1)) were analyzed. The study suggests that the by-product formation when carbon is limited is explained by a set of upregulated genes coupled to the shikimate pathway. The genes ydiB, aroD, and ydiN were strongly induced only in carbon-limited W3110.shik1. Compared to W3110, the 1 g(2)-fold changes were: 6.25 (ydiB), 3.93 (aroD), and 8.18 (ydiN). In addition, the transcriptome analysis revealed a large change in the gene expression when comparing phosphate-limited conditions to carbon-limited, which to a large part could be explained by anabolic-catabolic uncoupling, which is present under phosphate-limited but not under carbon-limited conditions. Interestingly, there was also a larger difference between the two strains under carbon-limited conditions than under phosphate-limited. The reason for this difference is interpreted as a starvation for aromatic amino acids under carbon-limited conditions, which is relieved under phosphate-limited conditions due to an upregulation of aroK and aroA (Johansson L. and Liden G., J. Biotechnol. In Press, Corrected Proof, Available online 17 May 2006).
But currently, there have been no reports of attenuating expression of the ydiN gene or the ydiB gene or the combination thereof for the purpose of producing L-amino acids.